Our broad long-term objectives are to elucidate mechanisms that cause loss of growth inhibition by TGF- b in endometna adenocarcinoma (ECA) and to define hormone regulation of TGF-b through stromal/epithelial interactions in the endometrium. ECA is induced by estrogenic (E2) agents causing hyperproliferation of uterine epithelial cells (UtE). Progesterone (Pg) is therapeutic due its growth inhibitory effect. TGF-b-mediated growth inhibition is transduced by two cooperating receptors (RI, RII) and the downstream signaling/transcription factors, Smad2/3,that activate genes that block cell cycle progression, such as the cyclin-dependent kinase inhibitor, p27kip1 We have shown that UtE isolated from all grades of ECA escape negative growth control by TGF-b by incurring multiple defects in the TGF-b response pathway including, loss of: TGF-b RH, activated Smad2, and p27kip1. Moreover, in complex hyperplasia (CH), the precursor to ECA, these proteins are already decreased. Thus, disruption of TGF-b action occurs early in endometrial carcinogenesis, providing an opportunity to understand molecular events leading to dysregulated growth. We will use primary cultures of normal, CH, and ECA UtE and co-cultures with stromal cells (UtS), which unlike ECA cell lines, retain many in vivo differentiation characteristics. Specific Aim 1, will determine the molecular mechanisms causing TGF-b receptor downregulation (e.g., transcriptional, translational) and test for defective Smad2/3 signaling using a TGF-b-promoter-responsive reporter assay. We will try to regain TGF-b function by transient transfection of RII cDNA into ECA UtE. Specific Aim 2 will test the hypothesis that loss of p27kip1 in ECA is by degradation via ubiquitin-proteasome pathway, which is E2-dnven directly through a MAPkinase via the Ras/MAPK/ERK1 pathway and that TGF-b normally prevents p27 degradation. Tissue/celilysates, inhibitors of proteasomes and MAPK, and immuno-analytical techniques will be used. Using single cell and co-cultures, we show that UtS from normal but not malignant endometnum mediates Pg-induced growth inhibition of normal UtE. Specific Aim 3 will test the hypotheses that UtS paracrine-mediates Pg-induced growth inhibition of UtE by release of TGF-b in response to Pg. We will use co-cultures of normal and "malignant" UtS and UtE and novel in vivo chimeric tissue recombinants composed of UtS from both, Pg receptor knock-out (PRKO) and wild-type mice and human UtE, that are hormonally manipulated as transplants in nude mice and UtE then analyzed for growth. These studies should elucidate molecular mechanisms of endometrial carcinogenesis, hormonal (dys)regulation of endometrial growth, and identify targets for prevention and therapeutic intervention.